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Papers by Tonya Nichols

Research Priority: Protocols for efficient production, certification, and distribution of pure to... more Research Priority: Protocols for efficient production, certification, and distribution of pure toxins and standards of consistent, high quality. Rapid and sensitive detection of cyanotoxins is an important goal for mitigating potential risks posed by HABs. Sensitivity and specificity of analytical methods for identifying the different classes of cyanotoxins and for accurate quantification depend on the availability of high quality toxin standards. The workgroup expressed concern that some published research was not as reliable as it might be because of reliance on in-house purification of toxins or use of commercial material that was later shown to be of poor quality. Standardized protocols are needed for the efficient production, certification, and distribution of pure toxins and standards. Commercial standards for cylindrospermopsin, some microcystins, nodularin, anatoxin-a, and saxitoxins are available (Table 1). However, concern regarding the reliability of some of the non-certified commercial standards has been expressed. Larger (mg) quantities of some cyanotoxins are available from certain research labs involved in cyanobacterial research, for example, the Australian Water Quality Centre (http://www.awqc.com.au/). Another concern is that recent regulations of high potency agents and toxins hamper the acquisition and storage of large toxin quantities that are needed for analytical methods development, validation, and, toxicological studies. Although safeguards are needed to prevent illicit uses of cyanotoxins, provisions for safe transportation and use in secure facilities are needed.

Journal of Microbiological Methods, Nov 1, 2016

Following a release of Bacillus anthracis spores into the environment, there is a potential for l... more Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14 spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

Microbe Magazine, Feb 1, 2008

Annals of Microbiology, Jul 8, 2014

Identifying virulent Bacillus anthracis within soil is a difficult task due to the number and div... more Identifying virulent Bacillus anthracis within soil is a difficult task due to the number and diversity of other organisms and impeding chemical constituents within soil. Regardless of the detection assay, the initial sample must be processed efficiently to ensure that debris, chemical components, and biological impurities do not obstruct downstream analysis. Soil sample processing protocols can be divided into two general types: indirect and direct. There are two requirements for successful indirect isolation of B. anthracis from soil samples: dissociate the spores from the soil particles and physically separate the free spores from the soil particles. Adding an aqueous carrier medium to a soil sample creates a sample slurry for easier manipulation. Centrifugation, high specific gravity separation, immunomagnetic separation, filtration, and settling have been used to physically separate spores from soil. Direct processing utilizes a soil sample without first separating the spores from the bulk sample and falls under two principal types: culturing on B. anthracis selective agar and bulk DNA extraction. Direct and indirect processing steps each have associated advantages and disadvantages. The objective of this review was to consolidate information acquired from previous research, focusing primarily on data gleaned in the last decade, on the processing of soils contaminated with B. anthracis. As shown in this review, an optimized soil-processing protocol with a known recovery rate and associated confidence intervals is needed. A reliable processing protocol would allow for multiple investigators and laboratories to produce high-quality, uniform results in the event of a B. anthracis release.

Journal of Applied Microbiology, Dec 27, 2012

Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium... more Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically detected C. parvum, C. hominis, Ent. faecium, B. anthracis and F. tularensis. The microarray was then evaluated with samples containing target and nontarget DNA from near-neighbour microorganisms , and tap water spiked with multiple organisms. Results demonstrated that the microarray consistently detected Ent. faecium, B. anthracis, F. tularensis and C. parvum when present in samples. Cryptosporidium hominis was only consistently detected through the use of shared probes between C. hominis and C. parvum. Conclusions: This study successfully developed and tested a microarray-based assay that can specifically detect faecal indicator bacteria and human pathogens in tap water. Significance and Impact of the Study: The use of indicator organisms has become a practical solution for monitoring for water quality. However, they do not always correlate well with the presence of many microbial pathogens, thus necessitating direct monitoring for most pathogens. This microarray can be used to simultaneously detect multiple organisms in a single sample. More importantly, it can provide occurrence information that may be used in assessing potential exposure risks to waterborne pathogens.

Environment International, Nov 1, 2014

Catastrophic incidents, such as natural disasters, terrorist attacks, and industrial accidents, c... more Catastrophic incidents, such as natural disasters, terrorist attacks, and industrial accidents, can occur suddenly and have high impact. However, they often occur at such a low frequency and in unpredictable locations that planning for the management of the consequences of a catastrophe can be difficult. For those catastrophes that result in the release of contaminants, the ability to analyze environmental samples is critical and contributes to the resilience of affected communities. Analyses of environmental samples are needed to make appropriate decisions about the course of action to restore the area affected by the contamination. Environmental samples range from soil, water, and air to vegetation, building materials, and debris. In addition, processes used to decontaminate any of these matrices may also generate wastewater and other materials that require analyses to determine the best course for proper disposal. This paper summarizes activities and programs the United States Environmental Protection Agency (USEPA) has implemented to ensure capability and capacity for the analysis of contaminated environmental samples following catastrophic incidents. USEPA's focus has been on building capability for a wide variety of contaminant classes and on ensuring national laboratory capacity for potential surges in the numbers of samples that could quickly exhaust the resources of local communities. USEPA's efforts have been designed to ensure a strong and resilient laboratory infrastructure in the United States to support communities as they respond to contamination incidents of any magnitude. The efforts include not only addressing technical issues related to the best-available methods for chemical, biological, and radiological contaminants, but also include addressing the challenges of coordination and administration of an efficient and effective response. Laboratory networks designed for responding to large scale contamination incidents can be sustained by applying their resources during incidents of lesser significance, for special projects, and for routine surveillance and monitoring as part of ongoing activities of the environmental laboratory community.

Journal of Parasitology, Oct 1, 2010

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in... more Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS-1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.

Springer eBooks, Mar 12, 2008

Rex A Pegram, Tonya Nichols, Stacey Etheridge, Andrew Humpage, Susan LeBlanc, Adam Love, Brett Ne... more Rex A Pegram, Tonya Nichols, Stacey Etheridge, Andrew Humpage, Susan LeBlanc, Adam Love, Brett Neilan, Stephan Pflugmacher, Maria Runnegar and Robert Thacke

Pathogens, Oct 24, 2020

Bacillus anthracis spores that are re-aerosolized from surface deposits after initial contaminati... more Bacillus anthracis spores that are re-aerosolized from surface deposits after initial contamination present significant health risks for personnel involved in decontamination. To model repeated exposure to low dose B. anthracis spores, three groups of seven rabbits were challenged with multiple low-doses of B. anthracis spores 5 days a week for 3 weeks. Mortality, body temperature, heart and respiration rates, hematology, C-reactive protein, bacteremia, and serum protective antigen were monitored for 21 days post-exposure after the last of multiple doses. All rabbits exposed to a mean daily dose of 2.91 × 10 2 colony forming units (CFU) survived and showed minimal physiological changes attributable to exposure. One of seven rabbits receiving a mean daily dose of 1.22 × 10 3 CFU died and four of seven receiving a mean daily dose of 1.17 × 10 4 CFU died. The LD 50 was calculated to be 8.1 × 10 3 CFU of accumulated dose. Rabbits that succumbed to the higher dose exhibited bacteremia and increases above baseline in heart rate, respiration rate, and body temperature. Two rabbits in the mean daily dose group of 1.17 × 10 4 CFU exhibited clinical signs of inhalation anthrax yet survived. This study provides a description of lethality, pathophysiology, and pathology in a controlled multiple low-dose inhalation exposure study of B. anthracis in the rabbit model. The data suggest that the accumulated dose is important in survival outcome and that a subset of rabbits may show clinical signs of disease but fully recover without therapeutic intervention

Environment International, Nov 1, 2014

Journal of Aerosol Science, Dec 1, 2017

This study experimentally assessed bacterial water-to-air partitioning coefficients resulting fro... more This study experimentally assessed bacterial water-to-air partitioning coefficients resulting from showerhead aerosolization of water contaminated with Brevundimonas diminuta or Pseudomonas aeruginosa, and estimated human exposure through inhalation. Dechlorinated tap water was spiked with two cell densities (10 9 and 10 10 CFU L −1) and cycled at three temperatures (10, 25, and 37 or 40°C) through a full-scale shower system. For reproducibility, spiked water concentrations were intentionally higher than those found in natural environments. Three types of samplers measured size distribution and viable concentrations throughout the system. Results indicate low levels of respirable bioaerosols were generated. The ratio of bacterial contaminant that was effectively aerosolized (bacterial water-to-air partitioning coefficient, PC bwa) was lowaveraging 1.13 × 10 −5 L m −3 for B. diminuta and 8.31 × 10 −6 L m −3 for P. aeruginosa. However, the respirable fraction of aerosolized organisms was high, averaging above 94% (in shower) and above 99% (downstream) for both organisms. This study found no significant difference in bioaerosol load for a forward facing versus reverse facing individual. Further, for the average hot shower (33-43°C) the total number of respirable bioaerosols is higher, but the observed culturability of those aerosolized cells is lower when compared to lower temperatures. Bacterial water to air partitioning coefficients were calculated to predict microbial air concentration and these empirical parameters may be used for assessing inhalation as a route of exposure to pathogens in contaminated waters.

Borrelia burgdorferi, the causative agent of Lyme disease, has been characterized as a microaerop... more Borrelia burgdorferi, the causative agent of Lyme disease, has been characterized as a microaerophilic spirochete. O2 consumption and utilization potentially yield reactive oxygen intermediates, such as superoxide, hydroxyl radicals, and hydrogen peroxide. This study investigated the expression of the sod gene, which encodes the only, identified oxidative defense mechanism in B. burgdorferi. Using primer extension analysis and RT-PCR, it was found that sod and secA are organized as a single transcriptional unit under the control of σ 70-like promoter upstream of the secA open reading frame. Generally, gene expression decreases with increased distance from the promoter; however, secA expression was observed to be relatively lower amounts than sod. Both genes were expressed in all phases of growth, with greatest expression observed in stationary phase. Because transcription of most sod genes is tightly regulated by iron concentration, the secA-sod gene expression was analyzed in borrelial cells grown in varying amounts of metal. Ironrestriction did not significantly affect growth nor did it affect transcription of secA or sod. Likewise, no major differences in transcription were observed with restricting or supplementing media with manganese. To test the possibility that the borrelial SOD may function with either iron or manganese as a cofactor as in cambialistic SODs, SOD activity was assayed and the highest activity was observed in increased manganese concentrations. Protein expression profiles of Sh-2-82 cultured in metal-defined media were investigated by one-and two-dimensional gel electrophoresis. Approximately 15 proteins were differentially expressed in response to metal concentration. iv ACKNOWLEDGMENTS There are many individuals who have played pivotal roles in helping me become the scientist and the person that I am today. As a graduate student at the University of Louisville, I was most influenced by my major professor, Dr. Faye Austin. I thank her for the opportunity to work in her laboratory on B. burgdorferi. Under her guidance, I received a good foundation in the difficult study of RNA genetics. From her, I have learned the value of attention to detail in experimental design, experimentation, and record-keeping, which will follow me throughout my scientific career. Dr. Uldis Streips has also been a key player in my success. I appreciate the time he has devoted to my project and me. I have good memories of our trips to WindRiver where scientific thoughts and ideas were exchanged in an informal, enjoyable manner. I also thank Dr. Robert Stout, Dr. Thomas Geoghegan, and Dr. Ronald Doyle, for their helpful suggestions during this project and their participation as committee members. Additionally, I thank Joan Gagel for her coordination efforts in the preparation of this dissertation. For her kindness and assistance, I would like to thank Jan Powars. There were many others who befriended me and supported me throughout my graduate study, and I am thankful for all of my friends at the University of Louisville. Most of all, I am grateful to my husband, Greg, and our two sons, Matthew and Joshua, for their love, support and understanding. Through a family effort, we have accomplished this goal together. v TABLE OF CONTENTS ABSTRACT….……………………………………………………………………………. iii ACKNOWLEDGMENTS…..……………………………………………………………... iv TABLE OF CONTENTS………………………………………………………………….. LIST OF TABLES….……………………………………………………………………… v viii LIST OF FIGURES………………………………………………………………………... ix LIST OF ABBREVIATIONS…………………………….…………………...…………... xi INTRODUCTION….……………………………………………………………………… I. Lyme disease….…………………………………………………………………. A. Epidemiology……………………………………………….…………….. B. Clinical features…………………………………………………………... C. Immunopathogenesis……………………………………………………… D. Treatment……………………………………………….………………… E. Vaccine……………………………………………………………………. II. Borrelia burgdorferi….…………….…………………………………………… A. Taxonomy ……………………………………………………………… B. Genetic organization……………………………………………………… C. Morphological features…………………………………………………… D. Metabolism…..…………………………………………………………… E. Virulence Factors….……………………………………………………… III. Transcriptional control in bacteria...…………………………………………… A. Organization of bacterial promoters……………………………………… B. Organization of borrelial promoters……………………………………… C. Processing of bacterial mRNA…..……………………………………….. IV. Bacterial responses to stress and environmental signals….…………………… A. Stationary phase….……………………………………………………….. B. Heat shock response….…………………………………………………… C. Borrelial heat shock response…………………….……………………….. D. Oxidative stress response….……………………………………………… E. Borrelial oxidative stress response……………….………………………. F. Metalloregulation of oxidative stress…..………………………………… V. SecA-dependent protein secretion……………………………………………… A. Overview of secretion mechanisms….…………………………………… B. Transcriptional organization and regulation of SecA…..………………… C. Secretory genes in B. burgdorferi…….…………………………………...

Pathogens, Jun 11, 2020

Credible dose-response relationships are needed to more accurately assess the risk posed by expos... more Credible dose-response relationships are needed to more accurately assess the risk posed by exposure to low-level Bacillus anthracis contamination during or following a release. To begin to fill this knowledge gap, New Zealand White rabbits were implanted with D70-PCT telemetry transmitters and subsequently aerosol challenged with average inhaled doses of 2.86 × 10 2 to 2.75 × 10 5 colony forming units (CFU) of B. anthracis spores. Rabbits exposed to a single inhaled dose at or above 2.54 × 10 4 CFU succumbed with dose-dependent time to death. Death was associated with increases above baseline in heart rate, respiration rate, and body temperature and all rabbits that died exhibited bacteremia at some point prior to death. Rabbits that inhaled doses of 2.06 × 10 3 CFU or lower survived to the end of the study and showed no or minimal adverse changes in the measured physiological responses in response to the challenge. Moreover, no bacteremia nor toxemia were observed in rabbits that survived to the end of the study. Overall, the data indicate that challenge doses of B. anthracis below the level sufficient to establish systemic infection do not produce observable physiological responses; however, doses that triggered a response resulted in death.

Geospatial Technology for Human Well-Being and Health

doi: 10.3389/fcimb.2012.00087 Inhalational anthrax(Ames aerosol) in naïve and vaccinated New Zeal... more doi: 10.3389/fcimb.2012.00087 Inhalational anthrax(Ames aerosol) in naïve and vaccinated New Zealand rabbits: characterizing the spread of bacteria from lung deposition to bacteremia

Advances in Experimental Medicine and Biology

The Cyanotoxins Workgroup was charged with the identification and prioritization of research need... more The Cyanotoxins Workgroup was charged with the identification and prioritization of research needs associated with: the identification of cyanotoxins; toxicokinetics and toxicodynamics of cyanotoxins; human susceptibility to the toxins; cyanobacterial genetics/ ...

Research Priority: Protocols for efficient production, certification, and distribution of pure to... more Research Priority: Protocols for efficient production, certification, and distribution of pure toxins and standards of consistent, high quality. Rapid and sensitive detection of cyanotoxins is an important goal for mitigating potential risks posed by HABs. Sensitivity and specificity of analytical methods for identifying the different classes of cyanotoxins and for accurate quantification depend on the availability of high quality toxin standards. The workgroup expressed concern that some published research was not as reliable as it might be because of reliance on in-house purification of toxins or use of commercial material that was later shown to be of poor quality. Standardized protocols are needed for the efficient production, certification, and distribution of pure toxins and standards. Commercial standards for cylindrospermopsin, some microcystins, nodularin, anatoxin-a, and saxitoxins are available (Table 1). However, concern regarding the reliability of some of the non-certified commercial standards has been expressed. Larger (mg) quantities of some cyanotoxins are available from certain research labs involved in cyanobacterial research, for example, the Australian Water Quality Centre (http://www.awqc.com.au/). Another concern is that recent regulations of high potency agents and toxins hamper the acquisition and storage of large toxin quantities that are needed for analytical methods development, validation, and, toxicological studies. Although safeguards are needed to prevent illicit uses of cyanotoxins, provisions for safe transportation and use in secure facilities are needed.

Journal of Microbiological Methods, Nov 1, 2016

Following a release of Bacillus anthracis spores into the environment, there is a potential for l... more Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14 spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

Microbe Magazine, Feb 1, 2008

Annals of Microbiology, Jul 8, 2014

Identifying virulent Bacillus anthracis within soil is a difficult task due to the number and div... more Identifying virulent Bacillus anthracis within soil is a difficult task due to the number and diversity of other organisms and impeding chemical constituents within soil. Regardless of the detection assay, the initial sample must be processed efficiently to ensure that debris, chemical components, and biological impurities do not obstruct downstream analysis. Soil sample processing protocols can be divided into two general types: indirect and direct. There are two requirements for successful indirect isolation of B. anthracis from soil samples: dissociate the spores from the soil particles and physically separate the free spores from the soil particles. Adding an aqueous carrier medium to a soil sample creates a sample slurry for easier manipulation. Centrifugation, high specific gravity separation, immunomagnetic separation, filtration, and settling have been used to physically separate spores from soil. Direct processing utilizes a soil sample without first separating the spores from the bulk sample and falls under two principal types: culturing on B. anthracis selective agar and bulk DNA extraction. Direct and indirect processing steps each have associated advantages and disadvantages. The objective of this review was to consolidate information acquired from previous research, focusing primarily on data gleaned in the last decade, on the processing of soils contaminated with B. anthracis. As shown in this review, an optimized soil-processing protocol with a known recovery rate and associated confidence intervals is needed. A reliable processing protocol would allow for multiple investigators and laboratories to produce high-quality, uniform results in the event of a B. anthracis release.

Journal of Applied Microbiology, Dec 27, 2012

Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium... more Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically detected C. parvum, C. hominis, Ent. faecium, B. anthracis and F. tularensis. The microarray was then evaluated with samples containing target and nontarget DNA from near-neighbour microorganisms , and tap water spiked with multiple organisms. Results demonstrated that the microarray consistently detected Ent. faecium, B. anthracis, F. tularensis and C. parvum when present in samples. Cryptosporidium hominis was only consistently detected through the use of shared probes between C. hominis and C. parvum. Conclusions: This study successfully developed and tested a microarray-based assay that can specifically detect faecal indicator bacteria and human pathogens in tap water. Significance and Impact of the Study: The use of indicator organisms has become a practical solution for monitoring for water quality. However, they do not always correlate well with the presence of many microbial pathogens, thus necessitating direct monitoring for most pathogens. This microarray can be used to simultaneously detect multiple organisms in a single sample. More importantly, it can provide occurrence information that may be used in assessing potential exposure risks to waterborne pathogens.

Environment International, Nov 1, 2014

Catastrophic incidents, such as natural disasters, terrorist attacks, and industrial accidents, c... more Catastrophic incidents, such as natural disasters, terrorist attacks, and industrial accidents, can occur suddenly and have high impact. However, they often occur at such a low frequency and in unpredictable locations that planning for the management of the consequences of a catastrophe can be difficult. For those catastrophes that result in the release of contaminants, the ability to analyze environmental samples is critical and contributes to the resilience of affected communities. Analyses of environmental samples are needed to make appropriate decisions about the course of action to restore the area affected by the contamination. Environmental samples range from soil, water, and air to vegetation, building materials, and debris. In addition, processes used to decontaminate any of these matrices may also generate wastewater and other materials that require analyses to determine the best course for proper disposal. This paper summarizes activities and programs the United States Environmental Protection Agency (USEPA) has implemented to ensure capability and capacity for the analysis of contaminated environmental samples following catastrophic incidents. USEPA's focus has been on building capability for a wide variety of contaminant classes and on ensuring national laboratory capacity for potential surges in the numbers of samples that could quickly exhaust the resources of local communities. USEPA's efforts have been designed to ensure a strong and resilient laboratory infrastructure in the United States to support communities as they respond to contamination incidents of any magnitude. The efforts include not only addressing technical issues related to the best-available methods for chemical, biological, and radiological contaminants, but also include addressing the challenges of coordination and administration of an efficient and effective response. Laboratory networks designed for responding to large scale contamination incidents can be sustained by applying their resources during incidents of lesser significance, for special projects, and for routine surveillance and monitoring as part of ongoing activities of the environmental laboratory community.

Journal of Parasitology, Oct 1, 2010

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in... more Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS-1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.

Springer eBooks, Mar 12, 2008

Rex A Pegram, Tonya Nichols, Stacey Etheridge, Andrew Humpage, Susan LeBlanc, Adam Love, Brett Ne... more Rex A Pegram, Tonya Nichols, Stacey Etheridge, Andrew Humpage, Susan LeBlanc, Adam Love, Brett Neilan, Stephan Pflugmacher, Maria Runnegar and Robert Thacke

Pathogens, Oct 24, 2020

Bacillus anthracis spores that are re-aerosolized from surface deposits after initial contaminati... more Bacillus anthracis spores that are re-aerosolized from surface deposits after initial contamination present significant health risks for personnel involved in decontamination. To model repeated exposure to low dose B. anthracis spores, three groups of seven rabbits were challenged with multiple low-doses of B. anthracis spores 5 days a week for 3 weeks. Mortality, body temperature, heart and respiration rates, hematology, C-reactive protein, bacteremia, and serum protective antigen were monitored for 21 days post-exposure after the last of multiple doses. All rabbits exposed to a mean daily dose of 2.91 × 10 2 colony forming units (CFU) survived and showed minimal physiological changes attributable to exposure. One of seven rabbits receiving a mean daily dose of 1.22 × 10 3 CFU died and four of seven receiving a mean daily dose of 1.17 × 10 4 CFU died. The LD 50 was calculated to be 8.1 × 10 3 CFU of accumulated dose. Rabbits that succumbed to the higher dose exhibited bacteremia and increases above baseline in heart rate, respiration rate, and body temperature. Two rabbits in the mean daily dose group of 1.17 × 10 4 CFU exhibited clinical signs of inhalation anthrax yet survived. This study provides a description of lethality, pathophysiology, and pathology in a controlled multiple low-dose inhalation exposure study of B. anthracis in the rabbit model. The data suggest that the accumulated dose is important in survival outcome and that a subset of rabbits may show clinical signs of disease but fully recover without therapeutic intervention

Environment International, Nov 1, 2014

Journal of Aerosol Science, Dec 1, 2017

This study experimentally assessed bacterial water-to-air partitioning coefficients resulting fro... more This study experimentally assessed bacterial water-to-air partitioning coefficients resulting from showerhead aerosolization of water contaminated with Brevundimonas diminuta or Pseudomonas aeruginosa, and estimated human exposure through inhalation. Dechlorinated tap water was spiked with two cell densities (10 9 and 10 10 CFU L −1) and cycled at three temperatures (10, 25, and 37 or 40°C) through a full-scale shower system. For reproducibility, spiked water concentrations were intentionally higher than those found in natural environments. Three types of samplers measured size distribution and viable concentrations throughout the system. Results indicate low levels of respirable bioaerosols were generated. The ratio of bacterial contaminant that was effectively aerosolized (bacterial water-to-air partitioning coefficient, PC bwa) was lowaveraging 1.13 × 10 −5 L m −3 for B. diminuta and 8.31 × 10 −6 L m −3 for P. aeruginosa. However, the respirable fraction of aerosolized organisms was high, averaging above 94% (in shower) and above 99% (downstream) for both organisms. This study found no significant difference in bioaerosol load for a forward facing versus reverse facing individual. Further, for the average hot shower (33-43°C) the total number of respirable bioaerosols is higher, but the observed culturability of those aerosolized cells is lower when compared to lower temperatures. Bacterial water to air partitioning coefficients were calculated to predict microbial air concentration and these empirical parameters may be used for assessing inhalation as a route of exposure to pathogens in contaminated waters.

Borrelia burgdorferi, the causative agent of Lyme disease, has been characterized as a microaerop... more Borrelia burgdorferi, the causative agent of Lyme disease, has been characterized as a microaerophilic spirochete. O2 consumption and utilization potentially yield reactive oxygen intermediates, such as superoxide, hydroxyl radicals, and hydrogen peroxide. This study investigated the expression of the sod gene, which encodes the only, identified oxidative defense mechanism in B. burgdorferi. Using primer extension analysis and RT-PCR, it was found that sod and secA are organized as a single transcriptional unit under the control of σ 70-like promoter upstream of the secA open reading frame. Generally, gene expression decreases with increased distance from the promoter; however, secA expression was observed to be relatively lower amounts than sod. Both genes were expressed in all phases of growth, with greatest expression observed in stationary phase. Because transcription of most sod genes is tightly regulated by iron concentration, the secA-sod gene expression was analyzed in borrelial cells grown in varying amounts of metal. Ironrestriction did not significantly affect growth nor did it affect transcription of secA or sod. Likewise, no major differences in transcription were observed with restricting or supplementing media with manganese. To test the possibility that the borrelial SOD may function with either iron or manganese as a cofactor as in cambialistic SODs, SOD activity was assayed and the highest activity was observed in increased manganese concentrations. Protein expression profiles of Sh-2-82 cultured in metal-defined media were investigated by one-and two-dimensional gel electrophoresis. Approximately 15 proteins were differentially expressed in response to metal concentration. iv ACKNOWLEDGMENTS There are many individuals who have played pivotal roles in helping me become the scientist and the person that I am today. As a graduate student at the University of Louisville, I was most influenced by my major professor, Dr. Faye Austin. I thank her for the opportunity to work in her laboratory on B. burgdorferi. Under her guidance, I received a good foundation in the difficult study of RNA genetics. From her, I have learned the value of attention to detail in experimental design, experimentation, and record-keeping, which will follow me throughout my scientific career. Dr. Uldis Streips has also been a key player in my success. I appreciate the time he has devoted to my project and me. I have good memories of our trips to WindRiver where scientific thoughts and ideas were exchanged in an informal, enjoyable manner. I also thank Dr. Robert Stout, Dr. Thomas Geoghegan, and Dr. Ronald Doyle, for their helpful suggestions during this project and their participation as committee members. Additionally, I thank Joan Gagel for her coordination efforts in the preparation of this dissertation. For her kindness and assistance, I would like to thank Jan Powars. There were many others who befriended me and supported me throughout my graduate study, and I am thankful for all of my friends at the University of Louisville. Most of all, I am grateful to my husband, Greg, and our two sons, Matthew and Joshua, for their love, support and understanding. Through a family effort, we have accomplished this goal together. v TABLE OF CONTENTS ABSTRACT….……………………………………………………………………………. iii ACKNOWLEDGMENTS…..……………………………………………………………... iv TABLE OF CONTENTS………………………………………………………………….. LIST OF TABLES….……………………………………………………………………… v viii LIST OF FIGURES………………………………………………………………………... ix LIST OF ABBREVIATIONS…………………………….…………………...…………... xi INTRODUCTION….……………………………………………………………………… I. Lyme disease….…………………………………………………………………. A. Epidemiology……………………………………………….…………….. B. Clinical features…………………………………………………………... C. Immunopathogenesis……………………………………………………… D. Treatment……………………………………………….………………… E. Vaccine……………………………………………………………………. II. Borrelia burgdorferi….…………….…………………………………………… A. Taxonomy ……………………………………………………………… B. Genetic organization……………………………………………………… C. Morphological features…………………………………………………… D. Metabolism…..…………………………………………………………… E. Virulence Factors….……………………………………………………… III. Transcriptional control in bacteria...…………………………………………… A. Organization of bacterial promoters……………………………………… B. Organization of borrelial promoters……………………………………… C. Processing of bacterial mRNA…..……………………………………….. IV. Bacterial responses to stress and environmental signals….…………………… A. Stationary phase….……………………………………………………….. B. Heat shock response….…………………………………………………… C. Borrelial heat shock response…………………….……………………….. D. Oxidative stress response….……………………………………………… E. Borrelial oxidative stress response……………….………………………. F. Metalloregulation of oxidative stress…..………………………………… V. SecA-dependent protein secretion……………………………………………… A. Overview of secretion mechanisms….…………………………………… B. Transcriptional organization and regulation of SecA…..………………… C. Secretory genes in B. burgdorferi…….…………………………………...

Pathogens, Jun 11, 2020

Credible dose-response relationships are needed to more accurately assess the risk posed by expos... more Credible dose-response relationships are needed to more accurately assess the risk posed by exposure to low-level Bacillus anthracis contamination during or following a release. To begin to fill this knowledge gap, New Zealand White rabbits were implanted with D70-PCT telemetry transmitters and subsequently aerosol challenged with average inhaled doses of 2.86 × 10 2 to 2.75 × 10 5 colony forming units (CFU) of B. anthracis spores. Rabbits exposed to a single inhaled dose at or above 2.54 × 10 4 CFU succumbed with dose-dependent time to death. Death was associated with increases above baseline in heart rate, respiration rate, and body temperature and all rabbits that died exhibited bacteremia at some point prior to death. Rabbits that inhaled doses of 2.06 × 10 3 CFU or lower survived to the end of the study and showed no or minimal adverse changes in the measured physiological responses in response to the challenge. Moreover, no bacteremia nor toxemia were observed in rabbits that survived to the end of the study. Overall, the data indicate that challenge doses of B. anthracis below the level sufficient to establish systemic infection do not produce observable physiological responses; however, doses that triggered a response resulted in death.

Geospatial Technology for Human Well-Being and Health

doi: 10.3389/fcimb.2012.00087 Inhalational anthrax(Ames aerosol) in naïve and vaccinated New Zeal... more doi: 10.3389/fcimb.2012.00087 Inhalational anthrax(Ames aerosol) in naïve and vaccinated New Zealand rabbits: characterizing the spread of bacteria from lung deposition to bacteremia

Advances in Experimental Medicine and Biology

The Cyanotoxins Workgroup was charged with the identification and prioritization of research need... more The Cyanotoxins Workgroup was charged with the identification and prioritization of research needs associated with: the identification of cyanotoxins; toxicokinetics and toxicodynamics of cyanotoxins; human susceptibility to the toxins; cyanobacterial genetics/ ...